Xueliang Li

The application of Genome-Scale Metabolic Network Models (GSMM) in fermentation optimization is hampered by challenges in differentiating viable from dead cells and parameter distortion induced by conventional detection methods. Using E. coli BL21(DE3) as the model organism, this study developed a flux analysis strategy that couples cell kinetics with GSMM. Key parameters were estimated using the gradient descent algorithm, thereby enabling precise prediction of viable cell concentration and glucose consumption dynamics. Integrating this with the Quadratic Programming-based parsimonious Flux Balance Analysis (QP-pFBA) algorithm, intracellular metabolic reaction fluxes were quantified. Results demonstrated that the model can effectively differentiate viable from dead cells; Batch D, adopting the gradient-increasing feeding strategy, achieved the maximum specific growth rate (μ_max) of 0.6457, the highest among the four batches. Moreover, key metabolic reaction fluxes were highly correlated with the feeding strategy. This framework forgoes specialized, high-cost equipment and offers robust cross-strain/process adaptability, thereby greatly advancing GSMM utility. It provides a powerful tool for precise fermentation control and accelerates the shift toward data-driven biomanufacturing.

The field of industrial biotechnology has shown an increasing interest in adopting digital twins for improving process productivity and management efficiency. Despite its potential benefits, digital-twin-based biomanufacturing has not been fully implemented. As a preliminary undertaking, we developed an open-source digital twin framework for cell culture. The core models of the digital twin were coded in C++ and compiled as a reusable Python library. A web-based, cloud-native HMI application that links the physical and virtual systems was developed. A microbioreactor digital twin system was implemented using the framework as a proof of concept. The system features a 3D-printed rocking platform that is customized to fit T25 flasks, enabling automated rocking rate and angle control and in-place optical cell density measurement. The digital twin was validated using Chinese Hamster Ovary (CHO) cells and was found to be able to predict the changes in cell density, glucose consumption, lactic acid production, and oxygen uptake rate (OUR). Finally, we performed a case study to demonstrate the system’s practical applicability in Advanced Process Control (APC) by constructing real-time glucose and lactic acid soft sensors, which are in turn used to alert the operator for manual media change or for automated feeding.

Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l⁻¹. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l⁻¹ was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.

The quality of foaming products (such as beer and shampoo) and the performance of industrial processes that harness foam (such as the froth flotation of minerals or the foam fractionation of proteins) depend upon foam stability. In this study, experiments are performed to study the effect of environmental humidity on the collapse of static foams. The dependency of the rate at which a foam collapses upon humidity is demonstrated, and we propose a hypothesis for bubble bursting due to Marangoni instability induced by nonuniform evaporation to help explain the dependency. This hypothesis is supported by direct experimental observations of the bursting process of isolated bubbles by high speed video recording and the thinning of isolated foam films under different values of humidity and temperature by microinterferometric methods.